The Effects And Mechanism Of Epithelial-to-mesenchymal Transition Caused By Angiotensin â…¡ In Human Hepatocytes In Vitro

BackgroundLiver fibrosis is an excessive wound healing response that occurs in most forms of chronic liver diseases and involves deposition of excess extracellular matrix leading to distorted architecture and culminating in cirrhosis. With ongoing liver damage, fibrosis may progress to cirrhosis, which is characterized by a distortion of the liver vasculature and architecture, predisposing to liver failure and primary liver cancer. So it has important social value and economic significance to explore the mechanism and preventive methods of liver fibrosis. The outcome of liver fibrosis is amphitropic, and some studies have reported that cirrhosis may also be reversed even in the advanced stage . There are no recognized effective drugs for the reversal of liver fibrosis. Therefore, the treatment of liver fibrosis is the focus and difficulty of the study for liver diseases.Recent reports and our previous studies have shown that intrahepatic renin-angiotensin system (RAS) is closely related with the occurrence and development of liver fibrosis. AngiotensinⅡ(AngⅡ), as an important effector molecule in the RAS , promotes the occurrence and development of liver fibrosis by activating hepatic stellate cells(HSC), portal myofibroblasts, and mesenchymal cells of bone marrow origin. HSC have dominated the field of liver fibrogenesis . Most of the existing research on AngⅡconcentrate on the mechanism of the activation and proliferation of the HSC.Unfortunately, despite of several discoveries pertaining to HSC activation and mechanisms of collagen deposition, no breakthroughs in substantial anti-fibrotic therapies have been developed in order to prevent the progression to cirrhosis and or reverse established fibrosis. Although other studies have also shown that hepatocytes may synthesize collagen, they believe that hepatocytes have an insignificant role in the perpetuation of liver fibrosis, and thus progression to cirrhosis.EMT (epithelial-mesenchymal-transition) is defined as a process in which epithelial cells loose their phenotypic characteristics and acquire typical feratures of mesenchymal cells such as fibroblasts.Previous studies mostly showed that EMT was indispensable in the process of embryonic development and the progression and migration of tumors. It has been found that three types of cells such as hepatocytes, bile duct epithelial cells and quiet HSC (Q-HSC) could obtain the phenotypes and gain the important function of fibroblasts via EMT, which is quite different with the previous concepts. A proposal to classify EMTs into three different biological subtypes based on the biological context in which they occur was discussed at a 2007 meeting on EMT in Poland and a subsequent meeting in 2008 at Cold Spring Harbor Laboratories. The EMTs that are associated with implantation, embryo formation, and organ development are organized to generate diverse cell types that share common mesenchymal phenotypes. The EMTs associated with wound healing, tissue regeneration, and organ fibrosis are of a second type. In these type 2 EMTs, the program begins as part of a repair-associated event that normally generates fibroblasts and other related cells in order to reconstruct tissues following trauma and inflammatory injury. In the setting of organ fibrosis, type 2 EMTs can continue to respond to ongoing inflammation, leading eventually to organ destruction. Tissue fibrosis is in essence an unabated form of wound healing due to persistent inflammation. Carcinoma cells undergoing a type 3 EMT may invade and metastasize and thereby generate the final, life-threatening manifestations of cancer progression.It shows that EMT is an important source of intrahepatic fibroblasts。Friedman SL, Chairman of American Association for the Study of Liver Diseases, believed that hepatocytes transdifferentiate into fibroblasts through EMT, which is an important finding in the study of liver fibrosis reported on Gastroenterology .Our study firstly investigates the effects and mechanism of EMT caused by AngⅡin human hepatocytes, and then evaluates the effects of AngⅡon ERK and RhoA-Rock signaling transduction pathways in hepatocytes.We hope that the investigation is helpful to understand the roles and mechanisms of RAS in liver fibrosis.ObjectiveTo investigate the effects and the mechanism of angiotensinⅡ(AngⅡ) on the induction of EMT in human hepatic immortal cell lines (HL-7702 cells) in Vitro.MethodsHL-7702 cells were cultured, then different following experiments would be carried out according to different experimental aims,Firstly, Cells were stimulated with different concentrations of AngⅡfor 72 hours, then the protein expression of Albumin,vimentin and E-cadherin was detected by Western Blot. It would be divided into control group,10-6mol/L AngⅡgroup, and 10-7mol/L AngⅡgroup.Secondly, Cells were stimulated with 10-7mol/L AngⅡfor different time period, and then the protein expression of Albumin,vimentin and E-cadherin was detected by Western Blot. It would be divided into 24hours control group,24hours AngⅡgroup, 48 hours control group,48 hours AngⅡgroup,72 hours control group and 72 hours AngⅡgroup.Thirdly, Cells were stimulated with 10-7mol/L AngⅡfor 72 hours, then F-actin combined with TRITC-Phalloidin and the protein expression of Albumin,vimentin and E-cadherin was detected by Immunofluorescence staining. It would be divided into control group and AngⅡgroup.Finally, The protein expression of Albumin,vimentin,E-cadherin and typeⅠcollagen among every group was detected by Western Blot,and the mRNA levels of typeⅠcollagen and snail-1 were detected by Realtime PCR. The groups were as follows, control group,TGF-β1 group,Angll group,Angll+irbesartan group,AngⅡ+Y27632 group,AngⅡ+PD98059 group. Cells were preincubated for 1 hour with inhibitors, such as irbesartan (inhibitor of Angll AT1 receptor),Y27632 (inhibitor of ROCK), PD98059 (inhibitor of ERK),all of them were at the concentration of 10-5mol/L, and then added to human Angll at the concentration of 10-7 mol/L for 72 hours, and TGF-β1 group was as a positive control group.ResultsFirstly, Cells were stimulated with 10-7 mol/L and 10-6 mol/L Angll for 72 hours. Compared with control group, AngⅡmarkedly increased vimentin expression and significantly decreased E-cadherin and albumin expression in a dose dependent manner. (P<0.01).Compared with 10-7 mol/L Angll group, the inductive effects of the 10-6 mol/L AngⅡgroup were not obivious (P<0.01);Secondly, the treatment of cells with human AngⅡfor 24,48 and 72 hours, at the dose of 10-7 mol/L,compared with the control group respectively, protein levels of Albumin,E-cadherin were significantly lower when stimulated with human Angll for 72 hours,and that of vimentin increased at 24 hours,and became highest at 48 hours.Thirdly, after 72 hours in culture, In control group, F-actin, binded with TRITC-phalloidin specially, was detected in the cell-cell junction with a peri-cell membrane distribution by Immunofluorescence staining, and AngⅡ-treated cells acquired a spindle-shaped morphology with polarization of the F-actin stress fibers throughout the cell. Compared with the control group, after stimulated with AngⅡfor 72 hours, cells acquired a spindle-shaped morphology and the protein expression of E-cadherin and albumin decreased and that of vimentin and type I collagen increased, detected by immunofluorescence staining.Finally, after 72 hours in culture, AngⅡand TGF-β1 increased protein expression of vimentin and typeⅠcollagen and lowered that of E-cadherin and albumin in cells, while those effects of AngⅡcould be inhibited by irbesartan, Y27632 and PD98059. AngⅡupregulated the mRNA level of typeⅠcollagen and snail-1 in cells, and the inhibitory effects of AngⅡcould be reversed by irbesartan, Y27632 and PD98059.ConclusionsAngⅡinduced EMT through ERK1/2 and Rho-ROCK pathway via AT-1 receptor in HL-7702 cells.